This project is focused on learning how promoters are opened to allow transcription to occur. The experiments involve studies of sigma factors, which are prime transcription regulators. The specific aims include: (1) To learn how energy is used to activate sigma 54 dependent promoters. (2) To learn the determinants on sigma 54 that mediate transcriptional activation and DNA-binding. (3) to learn how transcription levels are set at sigma 54 promoters, especially with regard to the role of downstream elements. (4) To learn the general mechanism by which open complexes are stabilized by sigma factors. The approach starts with the creation of libraries of mutants. These are screened to learn which are likely to be defective in specific functions of sigma 54. In vitro assays, including transcription, DNA-binding, protein binding and DNA melting are done to confirm these properties. Libraries of mutations are also created in DNA and the assays are repeated to learn the role of DNA sequence in activation. Mutant systems of greatest interest are studied intensively in vivo and in vitro to learn which parts of the protein and the DNA are most important for activation in vivo. Specific proposals for how activator and energy open the DNA are tested using these assays and also by probing for conformational changes in vitro. Selected comparisons are made between the sigma 54 and sigma 70 systems. The outcome is expected to be a description of the range of possibilities for how bacterial promoters may be opened for transcription. Opening is central to transcriptional control in all biological systems and is thus expected to enhance our understanding of the many diseases that involve defective control of transcription.